why my ligation can not work - (Feb/07/2006 )
i now try to ligase a 30bp fragment to the vector. first i anneal two single strand oligo ,heating to 95degree 5min and cool down to RT slowly followed by phenol/chloroform extraction and precipitation. then enzyme digest the annealed fragment and do the ligase. but i can not get any colony after transformation to the E.COli. how could i solve this problem?
thank you in advance
-rose9999_98-
How are you screening? Is it Blue/White selection? Sometimes, you can get read through small inserts resulting in blue colonies even though your ligation has worked?
I think we need more information though to give you any useful suggestions. Such as what controls you have used. What enzyme you are using etc..
M.
QUOTE (rose9999_98 @ Feb 8 2006, 03:49 PM)
i now try to ligase a 30bp fragment to the vector. first i anneal two single strand oligo ,heating to 95degree 5min and cool down to RT slowly followed by phenol/chloroform extraction and precipitation. then enzyme digest the annealed fragment and do the ligase. but i can not get any colony after transformation to the E.COli. how could i solve this problem?
thank you in advance
thank you in advance
-ML1975-
QUOTE (ML1975 @ Feb 8 2006, 06:54 PM)
How are you screening? Is it Blue/White selection? Sometimes, you can get read through small inserts resulting in blue colonies even though your ligation has worked?
I think we need more information though to give you any useful suggestions. Such as what controls you have used. What enzyme you are using etc..
M.
i now try to ligase a 30bp fragment to the vector. first i anneal two single strand oligo ,heating to 95degree 5min and cool down to RT slowly followed by phenol/chloroform extraction and precipitation. then enzyme digest the annealed fragment and do the ligase. but i can not get any colony after transformation to the E.COli. how could i solve this problem?
thank you in advance
I think we need more information though to give you any useful suggestions. Such as what controls you have used. What enzyme you are using etc..
M.
QUOTE (rose9999_98 @ Feb 8 2006, 03:49 PM)
i now try to ligase a 30bp fragment to the vector. first i anneal two single strand oligo ,heating to 95degree 5min and cool down to RT slowly followed by phenol/chloroform extraction and precipitation. then enzyme digest the annealed fragment and do the ligase. but i can not get any colony after transformation to the E.COli. how could i solve this problem?
thank you in advance
thank you very much for your reply. i used KpnI and BglII to digest the annealed oligo. when i used this two enzymes to digest my PCR products and did the ligation, it worked well always. i did not use Blue/white selection. i also used phosphotase to dephosphorylate my vector. in vector ligation, no colonies could be found indicated that no self ligation happaned. no problem with my transformation. i used a positive control and the control worked well. i think still is the problem of ligation or digestion enzyme can not work on such a short oligo.
-rose9999_98-